Process for producing nicotinic acid mononucleotide

ABSTRACT

Nicotinic acid mononucleotide is produced in significant amounts by fermentation by a process which comprises culturing a suitable microorganism belonging to the genus Brevibacterium, Corynebacterium, Arthrobacter or Micrococcus under aerobic conditions in an aqueous nutrient medium containing nicotinic acid or nicotinamide. Yields of greater than 2 mg./ml. are obtained in some cases.

United States Patent Nakayama et al.

[451 Mar. 21, 1972 PROCESS FOR PRODUCING NICOTINIC ACID MONONUCLEOTIDEInventors: Kiyoshi Nakayama, Sagamihara-shi; Haruo Tanaka, Machida-shi,both of Japan Assignee: Kyowa Hakko Kogyo, Ltd., Tokyo, Japan Filed:Mar. 25, 1969 Appl. No.: 810,349

Foreign Application Priority Data Mar. 29, 1968 Japan ..43/20057 US. Cl...19.5/28 N Int. Cl. .C12d 13/06 Field of Search 195/28 N [56]References Cited UNITED STATES PATENTS 3,368,947 2/1968 Nakayama et al.195/28 N Primary Examiner-Alvin E. Tanenholtz Attorney-Craig, Antonelliand Hill [57] ABSTRACT 14 Claims, No Drawings PROCESS FOR PRODUCINGNICOTINIC ACID MONONUCLEOTIDE coon This compound is useful as abiochemical reagent, as a starting material for the manufacture ofnicotinamide adenine dinucleotide and the like. Accordingly, processesfor its production have been sought in the art.

Among the prior art processes for the preparation of nicotinic acidmononucleotide, there have been reported the production thereof by anenzymatic reaction [Journal of Biological Chemistry, Vol. 236, p. 525(1961)] and its isolation from yeasts [Archives of Biochemistry andBiophysics, Vol. 82, p. 83 (1959)] and from Penicillium chrysogenum[Nature, Vol. 180, p. 1203 (1957)]. However, none of these processes issatisfactory for the industrial production of nicotinic acidmononucleotide.

Accordingly, one of the objects of the presentinvention is to provide animproved process for the production of nicotinic acid mononucleotidewhich overcomes the disadvantages and deficiencies ofthe prior artmethods.

Another object of the present invention is-to provide a process forproducing nicotinic acid mononucleotide which may be carried out in anefficacious and relatively simple manner.

A further object of the invention is to provide a process for producingnicotinic acid mononucleotide by fermentation which may be carried outadvantageously on an industrial scale at low cost to give a high yieldof product.

A still further object of the invention is to provide nicotinic acidmononucleotide.

These and other objects and advantages of the presentinvention willbecome apparent to those skilled in the art from a consideration of thefollowing specification and claims.

As the result of various investigations on a.process for producingnicotinic acid mononucleotide by the use of microorganisms. the presentinventors have found, in accordance with the present invention, thatsignificant amounts of nicotinic acid mononucleotide are produced andaccumulated in the resultant culture liquor if fermentation is carriedout in an aqueous nutrient medium to which is added nicotinic acid ornicotinamide. This finding has previously not been reported in the art.

As mentioned above, the detection or isolation of nicotinic acidmononucleotide by means of the prior art processes is difficult from anindustrial point of view because of the high costs of the startingmaterials, the low yields obtained, etc., however interesting theiracademic values may be. In comparison therewith, the present inventionis an extremely practical process and one that makes it possible toproduce nicotinic acid mononucleotide effortlessly in high yield at lowcost.

The characterizing features of this invention are, as noted above, toadd nicotinic acid or nicotinamide to the fermentation medium and to usebacteria belonging to a genus selected from the group consisting ofBrevibacterium, Corynebacteri' um,-Arthrobacter and Micrococcus as themicro-organism.

Either a synthetic culture medium or a natural nutrient medium issuitable for cultivation of the strains employed in the presentinvention as long as it contains the essential nutrients for the growthof the strain employed. Such nutrients are well known in the art andinclude substances such as a carbon source, a nitrogen source, inorganiccompounds and the like which are utilized by the micro-organism employedin appropriate amounts. Thus, as a carbon source, there may bementioned, by way of example, carbohydrates such as glucose, fructose,maltose, sucrose, starch, starch hydrolysate, molasses, mannose,glycerol, etc., or any other suitable carbon source such as organicacids, for example, acetic acid, lactic acid, pyruvic acid, etc. Thesesubstances may be used either singly or in mixtures of two or more.

As a nitrogen source, various kinds of inorganic or organic salts orcompounds, such as urea, liquid ammonia or ammonium salts such asammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate,ammonium phosphate, ammonium carbonate, etc., or natural substancescontaining nitrogen, such as cornsteep liquor, yeast extract, meatextract, peptone, fish meal, bouillon, casein hydrolysates (like NZ-Amine), casamino acid, fish solubles, rice bran extract, defattedsoybean cake, chrysalis hydrolysate, or various digestion substancesthereof, etc., may be employed. Again, these substances may be usedeither singly or in combinations of two or more.

Inorganic compounds which may beadded to the culture medium includemagnesium sulfate, sodium phosphate, potassium dihydrogen phosphate,potassium monohydrogen phosphate, ferrous sulfate, manganese chloride,calcium chloride, sodium chloride, zinc sulfate, manganese sulfate,calcium carbonate, etc.

If strains having specific nutritional growth requirements are employed,the substances needed to satisfy these requirements should, of course,be added to the culture medium. These include substances such as aminoacids, vitamins such as thiamine, cobalamin, etc., or biotin.

in accordance with this invention, nicotinic acid or nicotinamide isadded to the nutrient medium at the initiation of or during the sourceof fermentation, all at one time or in termittently. The nicotinic acidand nicotinamide can be added to the medium in the form of nontoxicsalts thereof such as the sodium salts, potassium salts, sulfates,hydrochlorides, ascorbate, etc. It is to be understood that suchequivalent additives fall within the scope of the present invention; Theamount of additive to be used can be varied in accordance with theparticular microorganism employed and the culturing conditions used.Generally, an amount of from O.l mg./ml. to mg./ml., preferably 1mg./ml. to 20 mg./ml., of additive nicotinic acid or nicotinamide isemployed.

Culturing is conducted under aerobic conditions, such as aerobic shakingof the culture or with aeration and agitation of a submerged culture, ata temperature of, for example, about 20 to 40 C. and at a pH of, forexample, about 4.0 to 9.5.

'After about 2 to 8 days of culturing under these conditions,

significant amounts of nicotinic acid mononucleotide are producedandaccumulated in the resultant culture liquor.

After the completion of culturing, the nicotinic acid mononucleotide isrecovered from the fermentation liquor by conventional means, such asion exchange resin treatment, extraction with solvents, precipitation,adsorption, chromatography, concentration or the like.

The following examples are given merely as illustrative of the presentinvention and are not to be considered as limiting. Unless otherwisenoted, the' percentages therein and throughout the application are byweight per liter of water. Exemplary microorganism strainsadvantageously employed in the present invention are described therein.

EXAMPLE 1 Brevibacterium ammoniagenes ATCC 6872 is used as the seedmicroorganism. This strain is cultured in a seed medium containing 2percent glucose, l percent peptone, 1 percent yeast extract, 0.3 percentNaCl, and 30 pg/l. of biotin at 30 C. for 24 hours. The resultant seedculture is inoculated in the ratio of percent (by volume) into afermentation medium having the following composition:

100 g. glucose 6 g. urea 10 g. yeast extract In preparing thefermentation medium, the above components are dissolved in 1 liter ofwater, and the pH thereofis adjusted to 8.0 with NaOl-l. Then, thefermentation medium is poured into individual flasks and is sterilizedin an autoclave at a pressure of l kg./cm. for 10 minutes.

After inoculation, ml. portions of the mixture ofseed and fermentationmedia are poured into 250 ml. conical flasks, respectively. Culturing isthen carried out with aerobic shaking at 30 C.

After 72 hours ofculturing. nicotinic acid amide is added to thefermentation liquor in an amount so as to give a concentration thereinof 2 mg./ml. Culturing is then further continued for 24 hours. As aresult, 2.3 mg./ml. of nicotinic acid mononucleotide is produced andaccumulated in the fermentation liquor.

The nicotinic acid mononucleotide is adsorbed on a polystyrene stronglybasic anion exchange resin [Dowex No. 1 (formic acid type)] and is theneluted with an aqueous solution ofammonium formate.

EXAMPLE 2 Culturing is conducted in the same manner as described inExample 1, except that nicotinic acid is used as the additive to themedium instead of nicotinamide. The amount of nicotinic acidmononucleotide produced in the resultant culture liquor is 2.3 mg./ml.

EXAMPLE 3 Culturing is conducted in the same manner as described inExample l, except that Corynebacterium sp. ATCC 2l084 is used as theseed microorganism instead of Brevibacterium ammoniagener. The amount ofnicotinic acid mononucleotide produced in the cultured liquor is l.7mg./ml.

EXAMPLE 4 Culturing is conducted in the same manner as described inExample 1, except that Arthrobacter sp. ATCC 21085 is used as the seedmicroorganism instead of Brevibacrerium ammoniagenes. The amount ofnicotinic acid mononucleotide produced in the cultured liquor is l.3mg./ml.

EXAMPLE 5 Micrococcus sadunensis ATCC l5932 is used as the seedmicroorganism. Culturing is conducted in the same manner and under thesame conditions as described in Example 1. As a result, the amount ofnicotinic acid mononucleotide produced and accumulated in thefermentation liquor is 0.4 mg./ml.

The invention being thus described. it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included herein.

What is claimed is: I I 1. A process for producing nlcotmic acidmononucleotide which comprises culturing a nicotinic acidmononucleotideproducing microorganism belonging to a genus selected fromthe group consisting of Brevibacterium, Corynebacterium, Arthrobacterand Micrococcus under aerobic conditions in an aqueous nutrient mediumcontaining nicotinic acid or nicotinamide, accumulating nicotinic acidmononucleotide in the resultant culture liquor, and recovering andisolating said nicotinic acid monocucleotide therefrom.

2. The process of claim 1, wherein said microorganism is Brevibacteriumammoniagenes.

3. The process of claim 1, wherein said microorganism is M icrococcussodonensis.

4. The process of claim 1, wherein the nicotinic acid or nicotinamide isadded to the medium at the initiation of culturing.

5. The process of claim 1, wherein the nicotinic acid or nicotinamide isadded to the medium after the initiation of culturing.

6. The process of claim 1, wherein the nicotinic acid or nicotinamide isadded to the medium in the form ofa sodium salt, potassium salt, sulfateor hydrochloride thereof.

7. The process of claim 1, wherein about I mg./ml. to 20 mg./ml. ofnicotinic acid is added to the medium.

8. The process of claim 1, wherein about 1 mg./ml. to 20 mg./ml. ofnicotinamide is added to the medium.

9. A process for producing nicotinic acid mononucleotide which comprisesculturing a nicotinic acid mononucleotideproducing microorganismbelonging to a genus selected from the group consisting ofBrevibacterium, Corynebacterium, Arthrobacter and Micrococcus at atemperature of about 20 to 40 C. and at a pH of about 4.0 to 9.5 underaerobic conditions in an aqueous nutrient medium containing nicotinicacid or nicotinamide, accumulating nicotinic acid mononucleotide in theresultant culture liquor, and recovering and isolating said nicotinicacid mononucleotide therefrom.

10. The process of claim 9, wherein about I mg./ml. to 20 mg./ml. ofnicotinic acid or nicotinamide is added to the medi- 11. The process ofclaim 10, wherein said microorganism is Brevibacrerium ammoniagenes ATCC6872.

12. The process of claim 10, wherein said microorganism isCorynebacterium sp. ATCC 2l084.

13. The process of claim 10, wherein said microorganism is Arthrobactersp. ATCC 21085.

14. The process of claim 10, wherein said microorganism is Micrococcussodonensis ATCC 15932.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTEON Patent N 3.650.898Dated March 21, 1972 fls) Kivoshi NAKAYAMA and Haruo TANAKA It iscertified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below: a

On the Title Page, left-hand column, line 5 -'Assignee: Kyowa HakkoKogyo, Ltd. Tokyo, Japan" should read:

--Assignee: Kyowa Hakko Kogyo Co. Ltd. Tokyo, Japan--- Signed and sealedthis 3rd day of October 1972.

(SEAL) Attest:

EDWARD MQFLETCI-ERJR. ROBERT GOTTSCHALK Attesting Officer Commissionerof Patents F ORM PO-105O (10-69) USCOMM-DC 6037G-P69 fl U.SGOVERNMENTPRINTING OFFICE: \969 0-366-336

2. The process of claim 1, wherein said microorganism is Brevibacteriumammoniagenes.
 3. The process of claim 1, wherein said microorganism isMicrococcus sodonensis.
 4. The process of claim 1, wherein the nicotinicacid or nicotinamide is added to the medium at the initiation ofculturing.
 5. The process of claim 1, wherein the nicotinic acid ornicotinamide is added to the medium after the initiation of culturing.6. The process of claim 1, wherein the nicotinic acid or nicotinamide isadded to the medium in the form of a sodium salt, potassium salt,sulfate or hydrochloride thereof.
 7. The process of claim 1, whereinabout 1 mg./ml. to 20 mg./ml. of nicotinic acid is added to the medium.8. The process of claim 1, wherein about 1 mg./ml. to 20 mg./ml. ofnicotinamide is added to the medium.
 9. A process for producingnicotinic acid mononucleotide which comprises culturing a nicotinic acidmononucleotide-producing microorganism belonging to a genus selectedfrom the group consisting of Brevibacterium, Corynebacterium,Arthrobacter and Micrococcus at a temperature of about 20* to 40* C. andat a pH of about 4.0 to 9.5 under aerobic conditions in an aqueousnutrient medium containing nicotinic acid or nicotinamide, accumulatingnicotinic acid mononucleotide in the resultant culture liquor, andrecovering and isolating said nicotinic acid mononucleotide therefrom.10. The process of claim 9, wherein about 1 mg./ml. to 20 mg./ml. ofnicotinic acid or nicotinamide is added to the medium.
 11. The processof claim 10, wherein said microorganism is Brevibacterium ammoniagenesATCC
 6872. 12. The process of claim 10, wherein said microorganism isCorynebacterium sp. ATCC
 21084. 13. The process of claim 10, whereinsaid microorganism is Arthrobacter sp. ATCC
 21085. 14. The process ofclaim 10, wherein said microorganism is Micrococcus sodonensis ATCC15932.